A system is provided for in vitro tracking of cancerous tissue over the course of the malignancy. The system provides a method for identifying the malignancy and for determining a patient""s prognosis. Further, the system provides for assessing a malignancy""s invasiveness, aggressiveness, growth rate, production of extracellular markers, possible side effects and for determining the efficacy on the malignancy of a given therapeutic regimen. The system also allows for generation of a therapeutic index, which serves as an indicator of a given therapy""s effectiveness against the malignancy as compared to its undesirable side effects, such as lethality to a patient""s normal cells.
Tracking a malignancy in a patient according to prior art methods is an inaccurate process which involves identification of the malignancy through techniques including biopsy and subsequent histological, biochemical, and immunochemical techniques and regularly monitoring the malignancy""s progression by invasive (i.e., biopsy) or noninvasive (i.e., x-ray, nuclear imaging, Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET)) methods. These methods are often expensive, inconvenient, painful and usually involve hospital visits and safety risks. It is, therefore, desirable to reduce a patient""s exposure to such methods. Furthermore, identification of a malignancy as a known variety of malignancy is often helpful in determining a suitable therapeutic approach and expected prognosis. However, even individually identifiable malignancies differ from patient-to-patient in their growth characteristics and in their responsiveness to treatment.
Determination of the growth rate, invasiveness and aggressiveness of a given malignancy is critical to prognosis and to the choice of therapies. A patient with a poor prognosis might be given a therapeutic regimen which might be more effective than another regimen but more risky to the patient. A patient with a better prognosis might be given a therapeutic regimen which is less aggressive and less risky to the patient, but which might not be as effective as often as a more dangerous therapy. Similarly, if a malignancy produces factors or creates conditions which cause a dangerous side effect, such as a thrombogenesis, the patient can be treated, preferably prophylactically, for the condition.
Current methodologies for determining growth rate, invasiveness, aggressiveness or which track the progression of a malignancy include biopsy and short-term culture, which can include drawing of blood or other bodily fluids, or semi- or non-invasive techniques such as x-ray and nuclear imaging. At any given time, a patient could be subject to multiple procedures, depending upon when the information is needed by the physician. Each procedure requires the presence of the patient and usually creates risk or pain. These procedures also can increase the stress level of the patient, which often is an exacerbating factor in cancer and associated prognoses. It is therefore, desirable to reduce the frequency of such procedures.
Identification of an effective therapeutic regimen is critically important to a patient. Often, once the malignancy is identified, a therapy is chosen based upon prior research on that type of malignancy and is not tailored to the sensitivities of the malignancy of a given patient. Often secondary therapies are needed because a first choice was ineffective. Valuable treatment time can be lost and a patient""s life can be threatened.
All active agents including chemotherapeutic active agents are subjected to rigorous testing as to efficacy and safety prior to approval for medical use in the United States. Methods of assessing efficacy have included elaborate investigations of large populations in double blind studies as to a given treatment method and/or active agent, with concomitant statistical interpretation of the resulting data, but these conclusions are inevitably generalized as to patient populations taken as a whole. In many pharmaceutical disciplines and particularly in the area of chemotherapy, however, the results of individual patient therapy may not comport with generalized dataxe2x80x94to the detriment of the individual patient. The need has been long recognized for a method of assessing the therapeutic potential of active agents, including but not limited to chemotherapeutic agents, for their efficacy as to a given individual patient, prior to the treatment of that patient. This need also applies to assessing the therapeutic potential as to radiation therapies, combined radiation/drug therapies and cellular immunotherapies.
Prior art assays already exist which expose malignant tissue of various types to a plurality of active agents, for the purpose of assessing the best choice for therapeutic administration. For example, in Kruczynski, A., et al., xe2x80x9cEvidence of a direct relationship between the increase in the in vitro passage number of human non-small-cell-lung cancer primocultures and their chemosensitivity,xe2x80x9d Anticancer Research, vol. 13, no. 2, pp. 507-513 (1993), chemosensitivity of non-small-cell-lung cancers was investigated in vivo grafts, in vitro primocultures and in commercially available long-term cancer cell lines. The increase in chemosensitivity was documented and correlated with morphological changes in the cells in question. Sometimes animal model malignant cells and/or established cell cultures are tested with prospective therapy agents, see for example Arnold, J. T., xe2x80x9cEvaluation of chemopreventive agents in different mechanistic classes using a rat tracheal epithelial cell culture transformation assay,xe2x80x9d Cancer Res., vol. 55, no. 3, pp. 537-543 (1995).
In vitro prior art techniques present the further shortcoming that assayed cells do not necessarily express the cellular markers they would express in vivo. This is regrettable because the determination of expression of certain secreted or cellular markers, secreted factors or tumor antigens or lack thereof can be useful for both identification and therapeutic purposes. For instance, members of the fibrinolytic system such as urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitors type 1 (PAI-1) are up-regulated in malignant brain tumors. See, e.g., Jasti S. Rao, et al., xe2x80x9cThe Fibrinolytic System in Human Brain Tumors: Association with Pathophysiological Conditions of Malignant Brain Tumors,xe2x80x9d Advances in Neuro-Oncology II, Kornblith P L, Walker M D (eds) Futura (1997). Other secreted factors such as xcex1-fetoprotein, carcinoembryonic antigen and transforming growth factors xcex1 and xcex2 have been found to be indicative of various cancers and/or cancer progression (see also, Singhal et al., xe2x80x9cElevated Plasma Osteopontin in Metastatic Breast Cancer Associated with Increased Tumor Burden and Decreased Survival,xe2x80x9d Clinical Cancer Research, vol. 3, 605-611, (April 1997); Kohno et al., xe2x80x9cComparative Studies of CAM 123-6 and Carcinoembryonic Antigen for the Serological Detection of Pulmonary Adenocarcinoma,xe2x80x9d Cancer Detection and Prevention, 21 (2): 124-128 (1997)). These examples are but a few of the many factors that may be used to identify diseased cells.
Cellular markers also include metastatic markers, indicative of metastatic potential, i.e., invasiveness and aggressiveness, which is relevant to the progression of a given malignancy and to a patient""s prognosis. First, markers indicating the invasiveness of a given malignancy indicate the ability of the malignancy to infiltrate and to destroy adjacent tissue. As an example, for epithelial malignancies, invasiveness markers are indicative of the ability of the malignancy to infiltrate beneath the epithelial basement membrane. Invasiveness markers can include the presence of proteolytic enzymes or angiogenic factors. A second category of metastatic marker indicates growth conditions of the malignancy. For instance, a malignancy could require for instance a prostate-specific factor for growth. Invasiveness and aggressiveness factors are often present in serum or in tissue culture media.
Relevant to a patient""s prognosis and, incidentally, to the identification of a malignancy is the presence of markers, cellular or secreted, which lead to complications beyond those involved with uncontrolled growth and invasion by a malignancy. For instance, secretion by the malignancy of thrombogenic substances by the malignancy can result in blood clotting, resulting in thrombophlebitis or other thrombotic events such as pulmonary thrombosis. Identification of a thrombotic potential indicates treatment (preferably prophylactically) with thrombolytic substances.
When a specific patient""s cells are used in vitro assays in typical prior art processes the cells are harvested (biopsied) and trypsinized (connective tissue digested with the enzyme trypsin) to yield a cell suspension purportedly suitable for conversion to the desired tissue culture form. The in vitro tissue culture cell collections which result from these techniques are generally plagued by their inability accurately to imitate the chemosensitivity or therapeutic sensitivity of the original tumor or other cell biopsy. These collections often do not express cellular markers in the same manner that they would in vivo. A need thus remains for a technique of tissue culture preparation which provides cell cultures, allowing identification of a malignancy, accurate tracking of the malignancy""s progress in a patient and therapy screening, in which, after simple preparation, the cell cultures react in a manner equivalent to their in vivo reactivity. The culture method would enable drug or chemotherapeutic agent, radiation therapy and/or cellular immunotherapy screening as to a particular patient for whom such screening is indicated.
A need also remains for a technique of tissue culture preparation which provides cell cultures for screening for expressed markers or factors where the cultured cells express the markers or factors in a manner indicative of their in vivo expression of the same. A further need also remains for a tissue culture preparation which allows for morphological study of the cells. Lastly, a need remains for a tissue culture system in which progression of an individual malignancy can be studied as indicative of the in vivo progression of the malignancy.
A comprehensive and integrated unified system for monitoring (i.e., identifying, tracking and analyzing) an individual patient""s malignancy through the duration of a malignancy as to a specific patient is provided. The method of the present invention allows for initial identification of a malignancy, identification of malignancy-specific cellular or secreted markers, identification of cellular or secreted markers indicative of complications, study of the invasiveness and aggressiveness of the malignancy, study of the growth rate of the malignancy, study of the effect of therapies on the malignancy as compared to control cells of the same patient (chemosensitivity versus toxicity) and the identification of a therapeutic index (i.e., the ratio of chemosensitivity:toxicity), study of tumor morphology and study of histological and cytochemical markers.
The method of the present invention includes the steps of collecting a tissue sample or specimen of a patient""s cells and separating the specimen into cohesive multicellular particulates (explants) of the tissue sample, rather than enzymatically digested cell suspensions or preparations. The cells are then grown as a tissue culture monolayer from the multicellular particulates to form a prime culture. A specimen can be taken from a patient at any relevant site, including but not limited to tissue, ascites or effusion fluid. Samples may also be taken from body fluid or exudates, as is appropriate. A tissue culture monolayer, designated as the prime culture, can be grown in any method known in the art for growing such a monolayer, for instance in tissue culture plates or flasks. If the malignant cells originate from solid tissue, however, the tissue must be subdivided into small pieces from which a tissue culture monolayer is then grown out.
Once a prime culture is established from a patient""s malignancy, the prime culture can be maintained without any treatments beside normal feedings and passage techniques, as indicative of the growth of the malignancy absent treatment. However, subcultures of the prime culture are prepared so that the prime culture is preferably left untreated, and the cells of the prime culture are not affected by any testing. However, either the prime culture or a subculture thereof can be propagated as a reference culture. The reference culture is a culture which is treated with therapies reflective of a patient""s actual treatments. For instance, if a patient is treated with a chemotherapeutic agent, the reference culture is treated with the same agent in the same concentration. The reference culture can be monitored genotypically or phenotypically to reflect actual progress of the malignancy in the patient. Treatment of the reference culture need not be limited to anticancer therapies, but can reflect all of a patient""s treatments. For instance, thrombolytic or anti-thrombogenic treatments can be applied to the reference culture to reflect a patient""s treatment. Subcultures of either the prime culture or the reference culture can be used for further analysis. Preferably, since the reference culture is indicative of the current state of a malignancy at a given time, subcultures of the reference culture are analyzed further. At various points in the passage of the control culture and the reference culture, aliquots of cells from those cultures can be stored cryogenically or otherwise.
The tissue sample technique of the present invention is also useful in assaying expression and/or secretion of various markers, factors or antigens present on or produced by the cultured cells. These assays can be used for diagnostic purposes for monitoring the applicability of certain candidate therapeutic or chemotherapeutic agents or for monitoring the progress of treatment of the cancer with those agents.
A method for identifying and monitoring progress of a malignancy in an individual patient is provided including the steps of inoculating cells from either the prime culture, the reference culture or a subculture of the prime culture or of the reference culture into a plurality of segregated sites; treating the plurality of sites with at least one treating means or therapy, followed by assessment of sensitivity of cells in the site to the treating means; collecting a specimen of a patient""s non-malignant cells; separating the non-malignant cells into cohesive multicellular particles; growing a tissue culture monolayer from the multicellular particulates of non-malignant cells to form a control culture; inoculating the control culture in a plurality of non-segregated sites; treating the plurality of segregated sites of the control culture with the same treating means as the segregated sites of the prime culture or a subculture thereof, followed by assessment of the sensitivity of the segregated cells of the control culture to the treating means; and comparing the sensitivity of the segregated cells of the prime culture or a subculture thereof with the sensitivity of the segregated cells of the control culture to the treating means. The assessments described above are calculations of the percentage or fraction of cells sensitive, or insensitive, to the treatment and the method further includes the step of creating a therapeutic index of a ratio of one of the percentage of or the fraction of sensitive cells or insensitive cells in the segregated cells of the control culture to one of the percentage of or the fraction of sensitive cells or insensitive cells in the segregated cells of the prime culture or subculture thereof.
Lastly, a method for treating a patient having a malignancy is provided, including the steps of: analyzing a patient""s cells prepared according to the above-described methods for malignancy-associated markers; determining a therapeutic regimen according to the results of the analysis; and treating a patient according to the regimen. The method can further include the step of treating one of either cells cultured as a subculture of the prime culture or cells of the prime culture according to the regimen as representative of the patient""s malignancy. Lastly, the method further includes determining a therapeutic index for each treating means as described above.
When applicable, cultures can be grown in a readable (scannable) plate and to determine percent confluence of the cells or any other parameter which can be determined in such a manner. The scanner can be operably linked with a computer or CPU to automatically input data into the computer or CPU. The computer or CPU can be programmed to automatically calculate a therapeutic index (or other relevant indices) based upon the data provided by the scanner. Alternatively, the data can be entered manually into the programmed computer or CPU to calculate the index.